1. Technical Field
This application relates generally to pharmacological compositions and methods for treating or reducing the risk of bacterial infection and, in particular, to compositions comprising an RNAIII-inhibiting peptide and an antimicrobial peptide and/or an antibiotic that is an aminoglycoside, beta-lactam, caphalosoprin or vancomycin.
2. Background of the Technology
Sepsis
Sepsis remains a leading cause of death, despite improvements in antimicrobial drugs and better supportive care. Sepsis is associated with systemic inflammation, circulatory failure, and multiple organ dysfunction syndrome (MODS). Both Gram-positive microbes, such as Staphylococcus aureus, and Gram-negative bacteria can cause sepsis. The incidence of sepsis is currently on the rise. Angus et al., Crit. Care Med. 29: 1303-10 (2001). Gram-negative bacteria release lipopolysaccharide (LPS), or endotoxin, from their outer membrane, which elicits septic shock. By contrast, some Gram-positive bacteria cause septic shock by the release of enterotoxins, 23 to 29 kDa polypeptides in the bacterial superantigen protein family, such as toxic shock syndrome toxin-1 (TSST-1), and exotoxins, such as pyrogenic exotoxin A. Exotoxins are soluble substances that alter the normal metabolism of host cells with deleterious effects on the host, while enterotoxins are exotoxins that are specific for intestinal cells. See De Kimpe et al., Proc. Nat'l Acad. Sci. USA 92: 10359-63 (1995); Kengatharan et al., J. Exp. Med. 188: 305-15 (1998); Llewelyn et al., Lancet Infect. Dis. 2: 56-162 (2002); Van Amersfoort et al., Clin. Microbiol. Rev. 16: 379-414 (2003).
Gram-positive bacteria cell wall components peptidoglygan (PG) and lipoteichoic acid (LTA) also have been shown to produce an inflammatory response. PG has a rigid structure and consists of repeating units of N-acetylglucosamine (β1-4)-linked to N-acetylmuramic acid. LTA molecules comprise repeating poly-(polyolphosphate) units and are highly variable for the presence of alditol groups that are modified with glycosil residues or D-alanine. LTA activates macrophages and polymorphonuclear leukocytes by binding to CD14, a surface receptor that also mediates responses to lipopolysaccharides. LTA acts synergistically with PG to release TNF-α and IL-6 and induce nitric oxide synthase (NOS) among other things, leading to circulatory failure, MODS and death. See De Kimpe (1995); Kengatharan (1998); Heumann et al., Infect. Immunol. 62: 2715-21 (1994); Scott et al., Infect. Immunol. 69: 875-88 (2001).
Quorum Sensing and RNAIII—Inhibiting Peptide
Recent studies have evidenced the importance of quorum-sensing in the pathology of bacterial species including Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus aureus. Quorum-sensing is a mechanism through which a bacterial population receives input from neighboring cells and elicits an appropriate response to enable itself to survive within the host. See Balaban et al., Science 280: 438-40 (1998); Miller et al., Cell 110: 303-14 (2002); Hentzer et al., EMBO J. 22: 3803-15 (2003); Korem et al., FEMS Microbiol. Lett. 223: 167-75 (2003). In Staphylococcus, quorum-sensing controls the expression of proteins implicated in bacterial virulence, including colonization, dissemination, and production of multiple toxins involved in disease promotion. Some of these virulence factors are enterotoxins and toxic-shock syndrome toxin-1 (TSST-1), which act as superantigens to cause over-stimulation of the host immune system, causing excessive release of cytokines and inducing the hyper-proliferation of T cells.
In a quorum-sensing system in S. aureus, the effector quorum-sensing molecule RNAIII-activating peptide (RAP) phosphorylates “target of RNAIII-activating protein” (TRAP), a 21 kDa protein that is highly conserved among staphylococci. TRAP phosphorylation promotes bacterial adhesion and the downstream production of a regulatory RNA molecule termed RNAIII, which is responsible for toxin synthesis. Balaban (1998); Balaban et al., J. Biol. Chem. 276: 2658-67 (2001). An antagonist of RAP called RNAIII-inhibiting peptide (RIP) inhibits the phosphorylation of TRAP and thereby strongly inhibits the downstream production of virulence factors, bacterial adhesion, biofilm formation, and infections in vivo. The mechanism of action of RIP is different from common antibiotics: instead of killing bacteria, RIP inhibits bacterial cell-cell communication, rendering the bacteria more vulnerable to host defense mechanisms. See Balaban (1998); Balaban et al., Peptides 21: 1301-11 (2000); Gov et al., Peptides 22: 1609-20 (2001); Balaban et al., J. Infect. Dis. 187:625-30 (2003); Cirioni et al., Circulation 108: 767-71 (2003); Ribeiro et al., Peptides 24: 1829-36 (2003); Giacometti et al., Antimicrob. Agents Chemother. 47: 1979-83 (2003); Balaban et al., Kidney Int. 23: 340-45 (2003); Balaban et al., Antimicrob. Agents Chemother. 48: 2544-50 (2004); Dell'Acqua et al., J. Infect. Dis. 190: 318-20 (2004).
Antimicrobial Peptides
Genetically encoded antimicrobial peptides are an important component of the innate immune response in most multi-cellular organisms that represents a first line of host defense against an array of microorganisms. Antimicrobial peptides have pleiotropic immunomodulatory functions and are endowed with direct antimicrobial activity and LTA/LPS-binding capacity. Antimicrobial peptides in circulating phagocytes contribute to the killing of engulfed microorganisms, and they act as a local defense mechanism in epithelial surfaces, protecting anatomical compartments from microbial invasion. See Cannon, Nature 328:478 (1987); Scott (1999); Hancock et al., Proc. Nat'l Acad. Sci. USA 97: 856-61 (2000); Giacometti et al., Gut 52: 874-78 (2003); Gough et al., Infect. Immun. 64:4922-27 (1996).
Cathelicidins are a family of related antimicrobial peptides that are produced as inactive precursors by several mammalian species on epithelial surfaces and within the granules of phagocytic cells. Cathelicidins exert a broad spectrum of antimicrobial activity against Gram-negative bacteria, Gram-positive bacteria and fungi with a wide overlap in specificity but also with significant differences in potency among antimicrobial peptide species. Like other antimicrobial peptides, cathelicidins bind LPS and neutralize its pro-inflammatory effects. See Zanetti et al., FEBS Lett. 374: 1-5 (1995); Zanetti et al., Curr. Pharm. Des. 8: 779-93 (2002); Zanetti et al., J. Leuk. Biol. 75: 39-48 (2004); Giacometti et al., Amer. J. Resp. Crit. Care Med. 169: 187-94 (2004).
Cathelicidins include BMAP-28, a peptide 27 amino acids in length with a primary amino sequence of GGLRSLGRKILRAWKKYGPIIVPIIRI-NH2 (SEQ ID NO: 1) and an amidated C-terminus. BAMP-28 kills antibiotic-resistant clinical isolates in vitro at submicromolar concentrations, and it retains a strong and broad activity spectrum in physiologic salt concentrations. BMAP-28 efficiently protects mice in vivo from lethal intraperitoneal infections in an acute peritonitis model. See Skerlavaj et al., J. Biol. Chem. 71: 28375-81 (1996); Benincasa et al., Peptides 24: 1723-31 (2003).
Conventional antibiotics are becoming less effective in dealing with the pathologies underlying sepsis and other serious diseases. For example, staphylococci currently are regarded as “super bugs” because of their capacity to acquire antibiotic resistance. Accordingly, there is an ongoing need for better compositions and methods to treat bacterial infections, particularly from Gram-positive bacteria such as Staphylococcus aureus. 